M. tuberculosis (Mtb) is one of the leading causes of death worldwide and claims millions of lives annually. Approximately 1.7 billion people worldwide are asymptomatically infected with the tubercle bacillus and constitute a major impediment to worldwide public health control measures. Previous work had shown that a point mutation (Arg515->His) in the 4.2 domain of RpoV, the principal sigma factor in Mycobacterium bovis, is attenuating. Using the yeast two-hybrid system, we have established that the 4.2 domain of virulent Mtb specifically interacts with a regulatory protein WhiB3. In contrast, the attenuated RpoV allele containing the single point mutation was unable to interact with WhiB3. We constructed a Mtb whiB3 mutant (deltawhiB3) and showed that it behaved identical to the wild-type strain with respect to its ability to replicate in mice and guinea pigs in vivo. Mice infected with AwhiB3 showed significantly longer survival times than mice infected with the wild type Mtb. In addition, the lungs of AwhiB3-infected mice appeared much less adversely affected. It is notable that this virulence gene would not have been detected using conventional screens such as signature tagged mutagenesis, which screens for mutants primarily defective in growth, and not virulence. Furthermore, we have shown that a whiB3 mutant of virulent M. bovis, in contrast to AwhiB3, was completely attenuated for growth in guinea pigs. Mtb contain seven WhiB homologues that show strong homology to proteins that are critical for sporulation in Streptomyces spp. We hypothesize that WhiB3 regulates the expression of mycobacterial components that modulate the host immune system. To better understand the mechanism of whiB3 in Mtb virulence, we will use electron paramagnetic resonance spectroscopy (EPR) to biochemically characterize the WhiB3 Fe-S cluster genes, identify genes under WhiB3 control, and characterize proteins that interact with the WhiB family. We will also demonstrate that WhiB3 is a DNA binding protein capable of activating transcription of specific target genes. We will study the in vivo expression of the whiB family and their role in virulence. These studies will characterize the WhiB family as potential targets for interventions that may abolish virulence, but not growth. These studies will also provide insight into understanding whether TB is an anomalous immunological reaction in response to the persistent bacilli, whether the bacilli themselves induce lethal immunopathology, or if it is a combination of both.